Atherosclerosis and other vascular diseases account for more deaths in the U.S. than any other cause. It is now well-understood that atherosclerosis is an inflammatory disorder whereby in response to injury and insult to the vasculature, growth factors and cytokines stimulate normal vascular smooth muscle cells (VSMC) to proliferate and secrete excessive extracellular matrix material. The resultant lesions cause disruptions in normal blood flow, platelet aggregation and activation, and eventually end-organ damage due to circulatory failure. Much research in the past two decades has identified signaling pathways in VSMC that lead to these proliferative and secretory changes. One pathway that normally blocks VSMC proliferation and secretory activity and promotes normal gene expression is the nitric oxide (NO)/cGMP pathway. Cyclic GMP activates a serine/threonine protein kinase, PKG-I, in vascular smooth muscle that leads to increases in normal contractile protein gene expression and suppresses proliferative and secretory activity. PKG-I thus is an important homeostatic control point whose activity is important for preventing excessive VSMC phenotypic modulation to the fibroproliferative state. Over the past few years, our laboratory has been interested in the mechanisms that control the expression of PKG-I in VSMC. Briefly, PKG-I expression is suppressed by inflammatory cytokines and growth factors that increase the fibroproliferative phenotype; restoration of PKG-I expression by transfection/adenoviral transduction restores the contractile phenotype of the VSMC. Hence, how inflammation controls PKG-I expression is critically important to understand in order to fully understand how these vascular disorders come about. During the previous funding period, we identified a pathway by which inflammatory cytokines regulate PKG-I expression. By inducing the expression of type II NO synthase (iNOS) in VSMC, cytokines/growth factors cause a persistent elevation in cGMP which directly causes down-regulation in the levels of PKG-I. This, in turn, is caused by cGMP-induced autophosphorylation of the PKG-Ia isoform and its ubiquitinylation. In this proposal, we will investigate the mechanisms by which PKG-Ia is ubiquitinylated in cultured VSMC, and determine whether this mechanism exists in vivo using wild- type and iNOS null mice to define the role of iNOS. The results of this proposed three-year study will shed new light on the mechanisms by which inflammation bring about vascular disorders.